Cotton plants with improved cotton fiber characteristics and method for producing cotton fibers from these cotton plants

ABSTRACT

Cotton plants of the genus Gossypium with improved cotton fiber characteristics are disclosed. The cotton plant has an expression cassette containing a gene coding for an enzyme selected from the group consisting of endoxyloglucan transferase, catalase and peroxidase so that the gene is expressed in cotton fiber cells to improve the cotton fiber characteristics. Also disclosed is a method for producing cotton fibers with improved fiber characteristics from these cotton plants.

This application claims priority under 35 U.S.C. §120 to and is adivisional application of U.S. application Ser. No. 09/347,669 filedJul. 5, 1999 now abandoned.

FIELD OF THE INVENTION

The present invention relates to cotton plants that produce cottonfibers with improved fiber characteristics such as increased fiberlength, increased fiber fineness and higher fiber strength, and it alsorelates to a method for producing cotton fibers from these cottonplants.

BACKGROUND OF THE INVENTION

Cotton fibers are usually produced by cultivating cotton plants of thegenus Gossypium and collecting the cotton fibers from the capsules(cotton bolls) formed on the cotton plants. There are many varieties ofcotton plant, from which cotton fibers with different fibercharacteristics can be obtained and used for various applicationsdepending on their fiber characteristics. Cotton fibers arecharacterized by various properties among which fiber length, fiberfineness and fiber strength are particularly important. Many effortshave been made so far to improve the characteristics of cotton fibers.Attempted improvements have been mainly focused on fiber length andfiber fineness. In particular, there has been a great demand for longerand finer cotton fibers. The variety of cotton plant known as Sea Islandis famous for desired fiber characteristics; however, this variety ofcotton plant exhibits a poor yield of cotton fibers and therefore theprice of Sea Island cotton fibers is very high. If highly yieldingcotton plants with cotton fiber characteristics equal to or better thanthose of Sea Island cotton can be produced, it will be a greatcontribution to industry.

The methods for improving the characteristics or yield of cotton fiberscan be roughly classified into the following three categories:

1. Variety Improvement by Cross Breeding

This method has been utilized most widely so far. At present, almost allthe cultivated varieties of cotton plant are bred by this method.However, much time is needed for this method and the degree ofvariability is limited, so that one cannot expect remarkableimprovements in the cotton fiber characteristics or in the yield ofcotton fibers.

2. Treatment with Plant Hormone

Plant hormones such as auxin, gibberellin, cytokinin and ethylene havebeen put to practical use widely for field crops or horticulturalproducts. The influence of plant hormones on the fiber characteristicsof cotton plants, particularly on the fiber elongation mechanism, isknown by gibberellin, auxin or brassinolide; however, no effect has beenfully confirmed yet, and it cannot be said that these plant hormones areeffective for practical use.

3. Variety Improvement by Gene Recombination Technique

In recent years, gene recombinant technique has made remarkableprogress, and several reports have been made on the successful varietyimprovement in certain kinds of plants (e.g., cotton, soybean, corn,tomato) by introduction and expression of a particular gene in theseplants to confer a desired genetic trait thereon. There have beendeveloped and put to practical use, for example, cotton plants withimproved insect resistance by introduction of a gene coding for BT toxin(i.e., insecticidal protein toxin produced by Bacillus thuringiensis) orcotton plants with improved herbicide (Glyphosate) resistance byintroduction of a gene coding for 5-enol-pyruvil-shikimic acid3-phosphate synthetase.

If a gene associated with cotton fiber formation and elongation can beintroduced into cotton plants and expressed in sufficient quantities, itwould become possible to make a remarkable improvement in thecharacteristics or yield of cotton fibers. Further, the introduction ofsuch a gene in anti-sense for makes it possible to suppress the actionof this gene. In other words, it is believed that the characteristicsand yield of cotton fibers can be controlled by introducing a geneassociated with fiber formation and elongation into cotton plants,followed by large-scale expression or suppression of the gene. Themethod using such a genetic engineering technique can be expected tofind wide applications, for example, more reliable control of fiberelongation and formation as compared with the conventional plantbreeding by cross-breeding and screening. For this purpose, it isrequired that the mechanisms of fiber elongation and formation areelucidated on the genetic level and some genes closely associated withthese mechanisms ark discovered and then actually expressed andregulated in the cotton fiber tissues to control the mechanisms of fiberelongation and formation.

At present, however, the knowledge of fiber elongation and formation inplants from the viewpoint of molecular biology is very limited. Althoughmany studies have been made on the elongation of plant cells, most ofthe control factors remain unknown and the control mechanisms have notyet been elucidated.

A cotton fiber is composed of a single cell that has been differentiatedfrom an epidermal cell of the seed coat, and it develops through fourstages, i.e., initiation, elongation, secondary cell wall thickening andmaturation stages. More specifically, the elongation of a cotton fiberbegins with that of an epidermal cell of the ovule just after floweringand the cotton fiber rapidly elongates and then completes elongation inabout 25 days after the flowering. After that, the fiber elongation isstopped, and a secondary cell wall is formed and grown throughmaturation to become a mature cotton fiber.

Some reports have been made on the isolation of such genes associatedwith the elongation and formation of cotton fibers. John et al. describethe isolation of E6 gene that is expressed preferentially in the cottonfiber tissues on the 15th and 24th days after flowering (see John, M. E.and Crow, L. J., Proc. Natl. Acad. Sci. USA, 89, 5769-5773 (1992)) or H6gene coding for a proline-rich protein that actively functions in theformation of secondary cell walls (see John, M. E. and Keller, G., PlantPhysiol., 108, 669-676 (1995)). John further examined the effects of E6gene on the cotton fiber characteristics by introduction of anti-senseE6 gene into cotton plants to reduce the expression level of endogenousE6 RNA (John, M. E., Plant Molecular Biology, 30, 297-306 (1996)). John,however, reported that although the expression level of E6 gene in fibertissues is reduced, fiber length, fiber strength and fiber fineness arenot significantly changed, and he concluded that E6 gene is not criticalto the normal development of cotton fibers. Song et al. identified acylcarrier protein (ACP) cDNA from cotton plants by differential displaymethod, which protein is specifically expressed in cotton fiber tissues(Song, P. and Allen, R. D., Biochimika et Biophysica Acta, 1351, 305-312(1997)). As the gene associated with the cellulose synthesis in cottonfibers, cDNA coding for a catalytic subunit of cellulose synthase wasisolated (Pear, J. R., Kawagoe, Y., et al., Proc. Natl. Acad Sci. USA,93, 12637-12642 (1996)). Kasukabe, one of the present inventors, and hiscoworkers have isolated and identified five genes from cotton plants bydifferential screening method and differential display method, whichgenes are specifically expressed at the cotton fiber elongation stage(assignees' own U.S. Pat. No. 5,880,100 and U.S. patent applicationsSer. Nos. 08/580,545, 08/867,484 and 09/262,653). Some genes associatedwith the elongation and formation of cotton fibers have already beenisolated from cotton plants; however, none have succeeded in modifyingthe fiber characteristics of cotton plants in practice.

The analysis of molecular mechanisms of plant cell wall construction ledto the isolation of endoxyloglucan transferase (EXGT) as an enzyme thatcatalyzes molecular grafting between polysaccharide cross-links in theplant cell wall matrix (Nishitani, K. and Tominaga, R., J. Biol. Chem.,267, 21058-21064 (1992)). Xyloglucans are polysaccharides thatcross-link individual cellulose microfibrils and play the main role inthe net work structure of the cell wall involved in the cell elongation.For this reason, the transfer of xyloglucan cross-links byendoxyloglucan transferase is considered one of the important processesin the cell elongation. Some genes coding for endoxyloglucan transferasehave been isolated from various plants including tomato (Lycopersiconesculentum) and mouse-ear cress (Arabidopsis thaliana) (Arrowsmith, D.A. and de Silva, J., Plant Mol. Biol., 28, 391-403 (1995); Okazawa, K.,Sato, Y., et al., J. Biol. Chem., 268, 25364-25368 (1993)), and furtherfrom cotton plants (Gossypium spp.) (Shimizu, Y., Aotsuka, S., et al.,Plant Cell Physiol., 38(3), 375-378 (1997)). There is, however, noreport that endoxyloglucan transferase genes isolated from variousplants or from cotton plants are actually introduced into cotton plantsand examined for the effects on the cotton fiber characteristics.

Most of the plant catalase species are localized on the microbodies andthey are detoxification enzymes that decompose metabolically producedtoxic H₂O₂ into water and oxygen. Their functions in plants have not yetbeen fully elucidated, although some reports have been made thatcatalase is associated with the low temperature tolerance and pathogenresistance of plants (Sanchez-Casas, P. and Klessing, D. F., PlantPhysiol., 106, 1675-1679 (1994); Prasad, T. K., Anderson, M. D., et al.,Plant Cell, 6, 65-74 (1994)). There is, however, no report on therelationship between catalase and fiber characteristics of cottonplants.

Peroxidase is an enzyme that catalyzes the following oxidative reactionby hydrogen peroxide: H₂O₂+AH₂→2H₂O+A

Peroxidase genes have been isolated from various plants including tomato(Lycopersicon esculentum) and horseradish (Armoracia rusticana)(Roberts, E., Kolattukudy, P. E., Mol. Gen. Genet., 217, 223-232 (1987);Fujiyama, K., Tekemura, H., et al., Eur. J. Biochem., 173, 681-687(1988)). Peroxidase is one of the cell wall enzymes and it existsthrough ionic bonding or covalent bonding in cell walls. One of thefunctions of peroxidase is the formation of lignin in the secondarycomponents of cell walls. Peroxidase is considered to catalyze, in thepresence of hydrogen peroxide, the reaction of forming dehydrogenationpolymeric products from the lignin components (e.g., ferulic acid,p-coumaric acid) produced through shikimic acid pathway or cinnamic acidpathway (Gross, G. G. et al., Planta, 136, 271 (1977)). Another reportdescribes that peroxidase is associated with the formation ofintermolecular cross-links by the reaction of the respective tyrosineresidues of two extensin molecules as the structural proteins of cellwalls (Lamport, D. T. A., MSU-DOE Plant Research Laboratory 7th AnnualReport, p. 65 (1982)). With respect to the relationship betweenperoxidase and cotton fibers, Rao et al. examined the peroxidaseactivity during the development of cotton fibers and showed that theperoxidase activity is lower in the cotton fiber elongation butsignificantly higher in the secondary wall thickening (Rao, N. R.,Naithani, S. C., et al., Z. Pflanzenphysiol. Bd., 106, 157-165 (1982)).These results suggest a possibility that peroxidase is involved in therigidifying of cotton fiber cell walls. John et al. confirmed that fiberstrength can be increased by introduction of a peroxidase gene intocotton plants and over-expression of the gene in the cotton fibertissues (WO95/08914); however, they failed to obtain significant resultson the fiber length or micronaire (i.e., fiber fineness).

As described above, some genes associated with the elongation andformation of cotton fibers have already been isolated; however, it seemsto be the most important to actually introduce these genes into cottonplants to make sure of their effects on the level of practical use.

SUMMARY OF THE INVENTION

Under these circumstances, the present inventors have extensivelystudied the mechanisms of fiber elongation and formation in cottonplants from the viewpoints of molecular biology to improve thecharacteristics of cotton fibers. As a result, they have found that thispurpose can be attained by introducing a gene coding for endoxyloglucantransferase, which is deeply associated with the cell elongation andgreatly expressed in the cotton fibers and ovule tissues at the cottonfiber elongation stage, or a gene coding for catalase or peroxidase,which is a hydrogen peroxide eliminating enzyme, into cotton plants andover-expressing these genes in the cotton fiber cells, therebycompleting the present invention.

Thus, the present invention provides a cotton plant of the genusGossypium with improved cotton fiber characteristics, comprising anexpression cassette containing a gene coding for an enzyme selected fromthe group consisting of endoxyloglucan transferase, catalase andperoxidase so that the gene is expressed in cotton fiber cells toimprove the cotton fiber characteristics; and cotton fibers and cottonseeds obtained from the cotton plant.

The present invention further provides a method for producing cottonfibers with improved fiber characteristics, and cotton fibers obtainedby this method. The method includes the steps of: transforming into acotton plant of the genus Gossypium, an expression cassette containing agene coding for an enzyme selected from the group consisting ofendoxyloglucan transferase, catalase and peroxidase so that the gene isexpressed in cotton fiber cells to improve the cotton fibercharacteristics; growing the cotton plant transformant to form cottonbolls; and collecting cotton fibers from the cotton bolls of the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the construction of plasmid pBI35S-22.

FIG. 2 is a diagram of the construction of plasmid pBIN35S-S.S-CAT.

FIG. 3 is a diagram of the construction of plasmid pBI35S-prxC1a.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have prepared cotton plant transformants that canexhibit the controlled expression of endoxyloglucan transferase,catalase or peroxidase gene by actually introducing each of these genes,together with an expression regulatory promoter, into cotton plants.They have evaluated the characteristics of cotton fibers obtained fromthese transformants and found that the fiber length, fiber strength andfiber fineness of these cotton fibers were significantly increased.

The term “endoxyloglucan transferase gene” as used herein refers to agene coding for the amino acid sequence of an enzyme that catalyzesmolecular grafting between polysaccharide cross-links in the plant cellwall matrix.

The term “catalase gene” as used herein refers to a gene coding for theamino acid sequence of an enzyme that catalyzes the decomposition ofhydrogen peroxide, H₂O₂+H₂O₂→O₂+2H₂O.

The term “peroxidase gene” as used herein refers to a gene coding forthe amino acid sequence of an enzyme that catalyzes the reaction,H₂O₂+AH₂→2H₂O+A.

The term “expression cassette” as used herein refers to a DNA fragmentcomprising a gene sequence coding for the amino acid sequence of anenzyme such as endoxyloglucan transferase, catalase or peroxidase, whichis operably linked to various regulatory elements that regulate theexpression of the gene sequence, such as promoter sequences and enhancersequences.

The term “cotton plant(s) that produces cotton fibers with improvedfiber characteristics” as used herein refers to a cotton plant(s) thatproduces excellent cotton fibers as compared with the conventionalcotton fibers with respect to fiber characteristics such as fiberlength, fiber strength and fiber fineness; for example, Coker 312(Gossypium hirsutum) as a typical variety of cotton plant can berendered ability to produce cotton fibers with increased fiber length,fiber fineness and/or fiber strength.

The term “cotton plant(s)” as used herein refers to those of the genusGossypium, examples of which are Gossypium hirsutum, Gossypiumbarbadense, Gossypium arboreum, Gossypium anomalum, Gossypiumarmourianum, Gossypium klotzchianum, Gossypium herbaceum and Gossypiumraimondii.

The phrase “gene hybridizes to . . . under stringent conditions” meansthat the gene may be modified DNA which can be obtained by selecting DNAthat hybridizes to DNA having the whole or part of the nucleotidesequence of SEQ ID NO: 1, 2 or 3 under stringent conditions. Moreparticularly, the endoxyloglucan transferase gene may hybridize to agene that has a nucleotide sequence extending from bps 32 to 901 of SEQID NO: 1, the catalase gene may hybridize to a gene that has anucleotide sequence extending from bps 57 to 1541 of SEQ ID NO: 2, andthe peroxidase gene may hybridize to a gene that has a nucleotidesequence extending from bps 1 to 1062 of SEQ ID NO: 3.

The term “stringent conditions” as used herein refers to those whichmake it possible that specific hybrids are formed but non-specifichybrids are not formed. The stringent conditions, although it isdifficult to define them by numerical values, are exemplified by thecase where modified DNA hybridizes to DNA having the whole or part ofthe nucleotide sequence of SEQ ID NO: 1, 2 or 3 under the hybridizationconditions, i.e., at 42° C. with a salt concentration of 6×SSC (0.9 MNaCl, 0.09 M trisodium citrate) or 6×SSPE (3M NaCl, 0.2 M NaH₂PO₄, 20 mMEDTA.2Na, pH 7.4) and then the hybrid remains undissociated even underthe washing conditions, i.e., at 42° C. with a salt concentration of0.5×SSC.

The following will describe with typical examples the isolation ofdesired genes (i.e., endoxyloglucan transferase, catalase and peroxidasegenes), the preparation of cotton plant transformants, and theevaluation of the characteristics of cotton fibers obtained from thetransformants.

1. Isolation of Cotton Endoxyloglucan Transferase Gene

(1) Construction of cDNA Library

From the cotton ovules on the 5th day after the flowering, poly(A)⁺ RNAis extracted by the ordinary method. Using the isolated poly(A)⁺ RNA asa template, cDNA is synthesized using oligo(dT) primers and reversetranscriptase, and then converted into double-stranded DNA by thepolymerase reaction. The double-stranded DNA is inserted into anappropriate vector, which is then used for the transformation ofEscherichia coli (hereinafter referred to as E. coli) or other hostcells to prepare a cDNA library.

The poly(A)⁺ RNA isolation and cDNA synthesis may also be carried outusing a commercially available cDNA cloning kit. Many kinds of vectorswhich can be used for library preparation are commercially available,such as λZAPII, λgt10 and λgt11. The host cell which can be used fortransformation may include E. coli XL-1 Blue, E. coli XL-1 Blue MRF^(v)and E. coli SURE.

(2) Screening of Desired Genes by Differential Screening Method

The phage plaque pattern of the cDNA library prepared by the abovemethod in section 1(1) is replicated onto two filters, which arehybridized with each of the ³²P-labelled cDNA probes prepared by thesame method as described in section 1(1) from the ovules on the 5th dayafter the flowering and from the ovules on the 25th day after theflowering. The cDNA corresponding to the desired gene can be selected bydetection of a positive hybridization signal only from the cDNA probeprepared from the ovules on the 5th day after the flowering.

The techniques necessary for the ordinary gene recombination such as RNAisolation, cDNA preparation, DNA digestion, ligation, transformation andhybridization are described in the instructional manuals of commerciallyavailable enzymes or agents used for the respective procedures orvarious text books (e.g., Molecular Cloning edited by Maniatis et al.,Cold Spring Harbor, 1989, and Current Protocols in Molecular Biologyedited by F. M. Ausubel et al., John Wiley & Sons, Inc., 1987).

The nucleotide sequence of the cloned cDNA can be determined by theMaxam-Gilbert method or the dideoxy chain termination method, each ofwhich can be carried out using a commercially available kit. Thenucleotide sequence may also be automatically determined with anauto-sequencer.

If the cDNA clone thus analyzed does not correspond to a full-lengthgene, a desired cDNA clone having such a full-length gene can beobtained by another plaque hybridization according to the ordinarymethod, or by the RACE (rapid amplification of cDNA ends) technique.

2. Isolation of Pea Catalase Gene

(1) Construction of cDNA Library

From the leaves of pea seedlings on the 10th day after the germination,poly(A)⁺ RNA is extracted by the ordinary method. Using the isolatedpoly(A)⁺ RNA as a template, cDNA is synthesized using oligo(dT) primersand reverse transcriptase, and then converted into double-stranded DNAby the polymerase reaction. The double-stranded DNA is inserted into anappropriate vector, which is then used for the transformation of E. colior other host cells to prepare a cDNA library.

The poly(A)⁺ RNA isolation and cDNA synthesis may also be carried outusing a commercially available cDNA cloning kit. Many kinds of vectorswhich can be used for library preparation are commercially available,such as λZAPII, λgt10 and λgt11. The host cell which can be used fortransformation may include E. coli XL-1 Blue, E. coli XL-1 Blue MRF^(v)and E. coli SURE.

(2) Screening of Desired Genes by Plaque Hybridization Method

The phage plaque pattern of the cDNA library prepared by the abovemethod in section 2(1) is replicated onto a filter, which is hybridizedwith a ³²P-labelled cDNA probe corresponding to the cotton catalase gene(Ni W., Turley R. B. Trelease R. N., Biochem. Biophys. Acta., 1049,219-222 (1990)). The cDNA corresponding to the desired gene can beselected by detection of a positive hybridization signal.

The techniques necessary for the ordinary gene recombination such as RNAisolation, cDNA preparation, DNA digestion, ligation, transformation andhybridization are described in the instructional manuals of commerciallyavailable enzymes or agents used for the respective procedures orvarious text books (e.g., Molecular Cloning edited by Maniatis et al.,Cold Spring Harbor, 1989, and Current Protocols in Molecular Biologyedited by F. M. Ausubel et al., John Wiley & Sons, Inc., 1987).

The nucleotide sequence of the cloned cDNA can be determined by theMaxam-Gilbert method or the dideoxy chain termination method, each ofwhich can be carried out using a commercially available kit. Thenucleotide sequence may also be automatically determined with anauto-sequencer.

If the cDNA thus analyzed does not correspond to a full-length gene, adesired cDNA clone having such a full-length gene can be obtained byanother plaque hybridization according to the ordinary method, or by theRACE (rapid amplification of cDNA ends) technique.

3. Isolation of Horseradish Peroxidase Gene

(1) Construction of cDNA Library

From the cultured cells of horseradish, poly(A)⁺ RNA is extracted by theordinary method. Using the isolated poly(A)⁺ RNA as a template, cDNA issynthesized using oligo(dT) primers and reverse transcriptase, and thenconverted into double-stranded DNA by the polymerase reaction. Thedouble-stranded DNA is inserted into an appropriate vector, which isthen used for the transformation of E. coli or other host cells toprepare a cDNA library.

The poly(A)⁺ RNA isolation and cDNA synthesis may also be carried outusing a commercially available cDNA cloning kit. Many kinds of vectorswhich can be used for library preparation are commercially available,such as λZAPII, λgt10 and λgt11. The host cell which can be used fortransformation may include E. coli XL-1 Blue, E. coli XL-1 Blue MRF^(v)and E. coli SURE.

(2) Screening of Desired Genes by Colony Hybridization Method

The colony pattern of the cDNA library prepared by the above method insection 3(1) is replicated onto a filter, which is hybridized with a³²P-labelled cDNA probe that can be synthesized by a commerciallyavailable DNA synthesizer from the amino acid sequence of horseradishperoxidase (Welinder, K. G., FEBS Lett., 72, 19-23 (1976)). The cDNAcorresponding to the desired gene can be selected by detection of apositive hybridization signal.

The techniques necessary for the ordinary gene recombination such as RNAisolation, cDNA preparation, DNA digestion, ligation, transformation andhybridization are described in the instructional manuals of commerciallyavailable enzymes or agents used for the respective procedures orvarious text books (e.g., Molecular Cloning edited by Maniatis et al.,Cold Spring Harbor, 1989, and Current Protocols in Molecular Biologyedited by F. M. Ausubel et al, John Wiley & Sons, Inc., 1987).

The nucleotide sequence of the cloned cDNA can be determined by theMaxam-Gilbert method or the dideoxy chain termination method, each ofwhich can be carried out using a commercially available kit. Thenucleotide sequence may also be automatically determined with anauto-sequencer.

If the cDNA clone thus analyzed does not correspond to a full-lengthgene, a desired cDNA clone having such a full-length gene can beobtained by another plaque hybridization according to the ordinarymethod, or by the RACE (rapid amplification of cDNA ends) technique.

4. Preparation of Cotton Plant Transformants

Any of the genes obtained by the above method may be ligated to anappropriate promoter, followed by introduction into cotton plants,making it possible to increase the content of a desired enzyme such asendoxyloglucan transferase, catalase or peroxidase. In contrast, atleast one part of the anti-sense strand (i.e., complementary sequence tothe coding sequence) of the above gene may be ligated in reversedirection to an appropriate promoter, followed by introduction intocotton plants and then expression of the so-called anti-sense RNA,making it possible to decrease the content of a desired enzyme.

The method for transformation of cotton plants may includeelectro-poration in which plasmids are introduced by treatment ofprotoplasts with electric pulses; fusion of small cells, cells orlysosomes with protoplasts; microinjection; polyethylene glycoltechnique; and particle gun technique.

With the use of a plant virus as a vector, a desired gene can also beintroduced into a cotton plant. A typical example of the plant viruswhich can be used is cauliflower mosaic virus (CaMV). The introductionof a desired gene is carried out, for example, as follows. First, avirus genome is inserted in a vector derived from E. coli or the like toprepare a recombinant, and a desired gene is inserted in the virusgenome. The virus genome thus modified is removed from the recombinantby restriction endonuclease and inoculated into a cotton plant to insertthe desired gene into the cotton plant (Hohn et al., Molecular Biologyof Plant Tumors, Academic Press, New York, 549-560 (1982), and U.S. Pat.No. 4,407,956].

Further, there is a technique using a Ti plasmid of Agrobacterium. Whena plant is infected with bacteria of the genus Agrobacterium, a part oftheir plasmid DNA is transferred to the plant genome. By making use ofsuch a property, a desired gene can also be introduced into a cottonplant. Upon inoculation; for example, Agrobacterium tumefaciens andAgrobacterium rhizogenes induce the formation of crown galls and theformation of hairy roots, respectively. Each of these bacteria has aplasmid designated “Ti-plasmid” or “Ri-plasmid” having T-DNA(transferred DNA) and vir region. The tumor formation is caused byintroduction of T-DNA into the genome of a plant, and then transcriptionand translation of an oncogene present in the T-DNA in the plant cells.The vir region per se is not transferred to the plant cells, but it isessential to the transfer of T-DNA. The vir region can also functioneven if it is on another plasmid different from the T-DNA containingplasmid (Nature, 303, 179 (1983)).

If a desired DNA is inserted in the T-DNA on the Ti- or Ri-plasmid, thedesired DNA can be introduced into the plant genome upon inoculation ofthe plant with these bacteria of the genus Agrobacterium. In this case,a portion inducing the formation of crown galls or hairy roots isremoved from the T-DNA of the Ti- or Ri-plasmid without deterioratingthe desired transfer function, and the plasmid thus obtained can be usedas a vector.

In the present invention, various other vectors can also be used, forexample, vectors such as pBI121 (Clontech), which are designated “binaryvectors”. A desired gene is ligated in sense or anti-sense direction toan appropriate promoter, which is then inserted in the binary vector,followed by introduction into a plant. These binary vectors have no virregion, and the bacteria of the genus Agrobacterium to be used forintroduction of these vectors are, therefore, required to containanother plasmid having vir region.

These vectors serve as a shuttle vector which can be amplified in E.coli as well as in the bacteria of the genus Agrobacterium. Accordingly,the recombination of Ti-plasmids can also be carried out with E. coli.These vectors have antibiotic-resistance genes, and the screening oftransformants can, therefore, be readily done in the transformation ofE. coli, bacteria of the genus Agrobacterium, or plants. These vectorsfurther have CaMV 35S promoter, and the gene inserted in these vectorscan, therefore, be introduced into the plant genome and then expressedunder constitutive control.

The introduction of a desired gene by Agrobacterium in cotton plantcells and the regeneration of whole cotton plants from the transformedcells can be achieved, for example, as follows:

According to the ordinary method, seeds of a cotton plant are sowed onStewart's seed germination media (Stewart's concentrate (detailedbelow), 0.75 g/l MgCl₂, 2.0 g/l Phytagel (Sigma), pH 6.8) andaseptically cultivated. The cotton seedlings are removed and hypocotylsare cut into small pieces. The desired gene is ligated to an appropriatepromoter and subcloned in a plasmid having kanamycin resistance gene.This clone is transformed into Agrobacterium and its culture is dilutedwith MSNH (MS basal salts (Sigma), 30 g/l glucose, pH 5.8). The dilutedAgrobacterium culture is used for the inoculation of the above hypocotylpieces. The inoculated hypocotyl pieces are blotted on sterile filterpaper to remove excess Agrobacterium solution, and placed on T₂ plates(MS basal salts (Sigma), 0.75 g/l MgCl₂, 0.1 mg/l 2,4-D, 0.5 mg/lKinetin, 30 g/l glucose, 2.0 g/l Phytagel (Sigma), pH 5.8), followed byincubation for 3 days. The hypocotyl pieces are transferred to MS2NK KCLplats (MS basal salts (Sigma), 0.75 g/l MgCl₂, 30 g/l glucose, 2 mg/lNAA, 0.1 mg/l kinetin, 2.0 g/l Phytagel (Sigma), 50 mg/l kanamycin, 500mg/l cefotaxime, pH 5.8) and incubated for 3 to 4 weeks to growcalluses. When the calluses are approximately 4 mm in size, they wereremoved from the hypocotyl and placed on fresh MS2NK KCL plates forfurther growth. When the calluses are sufficiently grown toapproximately 1 cm in size, they are placed in MSNH liquid mediacontaining kanamycin for embryo generation to begin the formation ofsuspension culture. The suspension culture is incubated until the cellsbecome light green in color with some slightly round cells visible inthe culture. The suspension culture containing fully grown cells isdiluted with MSNH, which is applied to MSK 50K plates (MS basal salts(Sigma), 0.75 g/l MgCl₂, 1.9 g/l KNO₃, 30 g/l glucose, 2.0 g/l Phytagel(Sigma), 50 mg/l kanamycin, pH 5.8) and spread over the media surface,followed by incubation for somatic embryo development. The incubation iscontinued until embryos reached at least 1 mm in size, they are placedon SA plates (Stewart's concentrate (detailed below), 20 g/l sucrose, 20g/l agar, pH 6.8) as embryo germination media. After continuedincubation for some weeks, the roots are trimmed when they are observed,and the embryos are transferred to SGA plates (Stewart's concentrate(detailed below), 5 g/l sucrose, 0.75 g/l MgCl₂, 5.0 g/l agar, 1.5 g/lPhytagel (Sigma), pH 6.8), followed by incubation which is furthercontinued until true leaves emerge. After emergence of the first trueleaves, the cotton young plantlets can be moved to pint sized canningjars containing SGA media. When these cotton young plantlets reachedapproximately 10 cm in height and have several true leaves, they can beset in appropriate pots and cultivated in a green house. Cotton seedsand cotton fibers can be obtained from the cotton plant transformantsthus regenerated.

From the transformants, DNA is extracted by the ordinary method anddigested with appropriate endonuclease. Southern hybridization using thedesired gene, which is the same as introduced into the transformants, asa probe makes it possible to determine whether transformation hasoccurred in the cotton plant.

In addition, from the transformants or non-transformants, RNA isextracted by the ordinary method, and a probe is prepared which has asense or anti-sense sequence of the desired gene, which is the same asintroduced into the transformants. Northern hybridization using theseprobes makes it possible to examine the degree of expression for thedesired gene.

5. Evaluation of Cotton Fiber Characterizations

From the regenerated transformants, those which have the desired geneintroduced therein and which express the desired gene are selected bySouthern hybridization or Northern hybridization. The transformants thusselected are cultivated, from which T1 seeds are obtained and thenexamined whether they are homozygotes or heterzygotes to select celllines having the desired gene surely introduced therein.

The cotton seeds of cell lines thus selected can be used for cultivationtest in a closed greenhouse or in a separated field. The cultivationtest is carried out as follows. The selected cotton seeds are sown bythe ordinary method, and the cotton plants are grown to come intoflower, followed by self-pollination. The subsequent opening of cottonbolls gives seed cotton, which is collected and subjected to machineginning by the ordinary method to give cotton fibers, which can beexamined for cotton fiber characteristics, for example, using the sortermethod, the pressley method, the stereometer method or the HVI (highvolume inspection machine) system method.

The present invention can attain the improvement of characteristics(e.g., fiber length, fiber fineness, fiber strength) of cotton fibers.With the use of cotton plants of the present invention, a novel varietyof cotton plant having a genetically fixed trait of producing cottonfibers with improved fiber characteristics in high yield.

The present invention will be further illustrated by the followingExamples; however, the present invention is not limited to theseExamples.

EXAMPLE 1 Cloning of Cotton Plant Endoxyloglucan Transferase Gene

1. Preparation of Poly(A)⁺ RNA

The cotton plant, Supima (Gossypium barbadense), cultivated in a fieldwas used as the test material. The ovules on the 5th day after theflowering and those on the 25th day after the flowering as a controlsample were collected. About 5 g of each of the ovules thus obtained wasimmediately frozen in liquid nitrogen and pulverized with a mortar inthe presence of liquid nitrogen. To the pulverized ovules was added 10ml of 0.2 M Tris-acetate buffer for extraction (5 M guanidinethiocyanate, 0.7% β-mercaptoethanol, 1% polyvinylpyrrolidone (M.W.,360,000), 0.62% N-lauroylsarcosine sodium salt, pH 8.5), and the mixturewas further pulverized with a polytron homogenizer (KINEMATICA) underice cooling for 2 minutes. At that time, β-mercaptoethanol andpolyvinylpyrrolidone were added to the buffer just before use. Thepulverized mixture was then centrifuged at 17,000×g for 20 minutes, andthe supernatant was collected.

The supernatant was filtered through a miracloth, and the filtrate wasgently overlaid on 1.5 ml of 5.7 M cesium chloride in a centrifuge tube,followed by centrifugation at 155,000×g for 20 hours at 20° C. Thesupernatant was discarded, and the precipitated RNA was then collected.The precipitate was dissolved in 3 ml of TE buffer (10 mM Tris-HCl, 1 mMEDTA.2Na, pH 8.0), to which a mixture of phenol, chloroform and isoamylalcohol (volume ratio, 25:24:1) was added at the same volume. Themixture was well agitated and then centrifuged at 17,000×g for 20minutes, and the upper aqueous layer was collected. To the aqueous layerobtained were added a 0.1-fold volume of 3 M sodium acetate (adjusted topH 6.2 by addition of gracious acetic acid) and a 2.5-fold volume ofethanol. The mixture was well agitated and allowed to stand withoutdisturbance at −20° C. overnight. The mixture was then centrifuged at17,000×g for 20 minutes, and the precipitate was washed with 70% ethanoland dried in vacuo.

The dry sample was dissolved in 500 μl of TE buffer to give a solutionof the whole RNA. This RNA solution was incubated at 65° C. for 5minutes and then immediately cooled on ice, to which 2×coupling buffer(10 mM Tris-HCl, 5 mM EDTA.2Na, 1M NaCl, 0.5% SDS, pH 7.5) was added atthe same volume. The mixture was overlaid on an oligo-dT cellulosecolumn (Clontech) which had been previously equilibrated withequilibration buffer (10 mM Tris-HCl, 5 mM EDTA.2Na, 0.5 M NaCl, 0.5%SDS, pH 7.5). The column was then washed with an about 10-fold volume ofthe same equilibration buffer as above, and the poly(A)⁺ RNA was elutedwith elution buffer (10 mM Tris-HCl, 5 mM EDTA.2Na, pH 7.5).

To the eluate obtained were added a 0.1-fold volume of the same 3Msodium acetate as above and a 2.5-fold volume of ethanol, and themixture was allowed to stand without disturbance at −70° C. The mixturewas then centrifuged at 10,000×g, and the precipitate was washed with70% ethanol and dried in vacuo. This dry sample was dissolved again in500 μl of TE buffer, and purification with an oligo-dT cellulose columnwas repeated as described above. The poly(A)⁺ RNA obtained from theovules on the 5th day after the flowering was used for the preparationof a cDNA library and a cDNA probe for differential screening, and thepoly(A)⁺ RNA obtained from the ovules on the 25th day after theflowering was used for the preparation of a cDNA probe for differentialscreening.

2. Preparation of cDNA Library at Fiber Elongation Stage

The preparation of a cDNA library was carried out with ZAP-cDNASynthesis Kit (Stratagene). The poly(A)⁺ RNA obtained from the ovules onthe 5th day after the flowering in section 1 was used as a template, anddouble-stranded cDNA was synthesized using oligo(dT) primers and reversetranscriptase according to the method of Gubler and Hoffman et al.(Gene, 25, 263-269 (1983)).

To both ends of the cDNA obtained were ligated EcoR I adaptors (havingXho I and Spe I sites in the inside), and the ligated DNA was digestedwith Xho I. The fragment was then ligated between the EcoR I and Xho Isites of λ phage vector, λZAPII arm, and the vector was packaged with anin vitro packaging kit (Stratagene, GIGAPACK Gold), followed byinjection into E. coli strain SURE (OD660=0.5), which afforded manyrecombinant λ phages serving as the cDNA library specific to the fiberelongation stage. This cDNA library had a size of 5.0×10⁶.

3. Preparation of Probes

The poly(A)⁺ RNA prepared from the ovules on the 5th or 25th day afterthe flowering was used as a template, and cDNA was synthesized usingoligo(dT) primers and reverse transcriptase M-MLV (Toyobo). After thesynthesis, alkali hydrolysis treatment was carried out to remove thepoly(A)⁺ RNA. The cDNA thus obtained was used as a template, and a³²P-labelled probe was prepared with Random Primed DNA Labeling Kit(USB).

The ³²P-labelled probes thus prepared by the cDNA on the 5th day afterthe flowering and by the cDNA on the 25th day after the flowering wereused as a probe for the fiber elongation stage and as a probe for thefiber non-elongation stage, respectively, for differential screening.

4. Screening of Genes Associated with Fiber Formation and Elongation

The above λ phages constituting the cDNA library from the ovules at the,fiber elongation stage were injected into E. coli, which was then grownon LB agar medium. About 50,000 plaques of λ phage DNA were replicatedon two nylon membranes (Hybond-N, Amersham).

The nylon membranes having replicated λ phage DNA were placed on afilter paper containing a solution for alkali denaturation (0.5 M NaOH,1.5 M NaCl) and allowed to stand for 4 minutes. The nylon membranes wereplaced on a filter paper containing a solution for neutralization (0.5 MTris-HCl, 1.5 M NaCl, pH 8.0) and allowed to stand for 5 minutes. Afterwashing with 2×SSC (0.3 M NaCl, 0.03 M trisodium citrate), thesemembranes were subjected to DNA fixation with Stratalinker (Stratagene).The nylon membranes thus treated for DNA fixation were prehybridized inhybridization buffer (50% formamide, 0.5% SDS, 6×SSPE (3M NaCl, 0.2 MNaH₂PO₄, 20 mM EDTA-2Na, pH 7.4), 5×Denhardt solution (0.1% Ficoll, 0.1%polyvinylpyrrolidone, 0.1% bovine serum albumin), 50 mg/l denaturedsalmon sperm DNA) at 42° C. for 3 hours, and the cDNA probes prepared insection 3 were added to the respective membranes, followed byhybridization at 42° C. for 20 hours. The membranes were then removedand washed with solutions each containing 2×SSC, 1×SSC, 0.5×SSC or0.1×SSC at 42° C. for 1 to 2 hours. These membranes were dried and thenallowed to adhere closely to X-ray films for exposure overnight.

As a result, 34 positive clones hybridized with the probe for the fiberelongation stage rather than for the fiber non-elongation stage wereselected. The particularly strongly hybridized clone was designated KC22and further analyzed.

From the phage DNA of KC22, plasmid clone pKC22 having a cDNA insert wasprepared with ZAP-cDNA Synthesis Kit (Stratagene) by the in vivoexcision method.

First, 200 μl of KC22-containing phage solution, 200 μl of E. coliXL1-Blue suspension and 1 μl of helper phage R408 suspension was mixedand then incubated at 37° C. for 15 minutes, followed by the addition of3 ml of 2×YT medium. A shaken culture was grown at 37° C. for 2 hoursand then treated at 70° C. for 20 minutes, followed by centrifugation at4000×g for 10 minutes, and the supernatant was collected. Then, 30 μl ofthe supernatant was mixed with 30 μl of E. coli SURE suspension, and themixture was incubated at 37° C. for 15 minutes and then inoculated onseveral microliters of LB agar medium containing 50 ppm ampicillin,followed by incubation at 37° C. overnight. The colony-forming E. colicontained the plasmid clone pKC22 having the cDNA insert.

The nucleotide sequence of the cDNA insert in the plasmid was determinedby the dideoxy chain termination method (Messing, Methods in Enzymol.,101, 20-78 (1983)). The resulting nucleotide sequence is shown in theSequence Listing, SEQ ID NO: 1.

The search on the homology between the nucleic acid sequences of thisgene and various known genes in the data base revealed that KC22 hashomology to the endoxyloglucan transferase genes isolated from otherplants such as red bean and tomato (Nishitani et al, J. Biol. Chem.,268, 25364-25368 (1993)) and to the meri-5 gene which is specificallyexpressed in an apical meristem of Arabidopsis (Medford, J. I., Elmer,J. S., and Klee, H. J., Plant Cell, 3, 359-370 (1991)).

EXAMPLE 2 Isolation of Pea Catalase Gene

The isolation of genes from a pea plant (Pisum sativum) was carried outby the method as described in Plant Molecular Biology, 17, 1263-1265(1991). The isolated pea catalase gene was designated CAT. Thenucleotide sequence of the gene is shown in Sequence Listing, SEQ ID NO:2.

EXAMPLE 3 Isolation of Horseradish Peroxidase Gene

The isolation of genes from horseradish (Armoracia rusticana) wascarried out by the method as described in Eur. J. Biochem., 173, 681-687(1988). The isolated horseradish peroxidase gene was designated prxC1a.The nucleotide sequence of the gene is shown in Sequence Listing, SEQ IDNO: 3.

EXAMPLE 4 Preparation of Cotton Plant Transformants

1. Construction of Plasmids

A plasmid with an expression cassette containing an endoxyloglucantransferase gene and a 35S promoter was prepared by the scheme as shownin FIG. 1. The nucleotide sequence of SEQ ID NO: 1 (KC22) was digestedwith Dra I so as to contain the open reading frame. The Dra I fragmentwas subcloned into the Sma I site of plasmid pUC19. This plasmid wasdigested with Sma I and Hin dIII, in which a Hin dIII/Xho I fragment ofa 35S promoter was subcloned. This clone was digested with Hin dIII andSac I, and the fragment was subcloned between the Hin dIII and Sac Isites of binary vector pBI101-Hm2 having kanamycin resistance gene(NPTII) and hygromycin resistance gene (HPT). This plasmid wasdesignated pBI35S-22 and transformed into E. coli JM109. The resultingE. coli JM109 transformant was designated E. coli JM109/pBI35S-22.

The plasmid with an expression cassette containing a catalase gene, asignal sequence and a 35S promoter was prepared by the scheme as shownin FIG. 2. In the expression cassette, the signal sequence (S.S.)corresponding to a signal peptide associated with the passage of anextracellular protein through the cell membrane was inserted between the35S promoter and the catalase gene. The signal sequence was amplifiedfrom the cotton plant cDNA library by PCR using primers with Xho I andBam HI restriction sites, respectively, according to the ordinarymethod, and subcloned in vector pRTL2. On the other hand, the nucleotidesequence of SEQ ID NO: 2 (CAT) was separated into the following threefragments: fragment 1 by digestion with Bam HI and Hin dIII; fragment 2by digestion with Hin dIII and Eco RI; and fragment 3 by digestion withEco RI and Xba I. Fragments 1 and 2 were ligated to the signal sequence,while fragment 3 was inserted between the 35S promoter (35S PROM) andthe terminator sequence (TRM). The fragment containing the signalsequence ligated to fragments 1 and 2 was digested with Xho I and EcoRI, and the fragment was subcloned between the 35S promoter and thefragment 3. This clone was digested with Pst I, and the fragment wassubcloned in binary vector pBIN19 having kanamycin resistance gene(nptII) with a Bam HI adapter. This plasmid was designatedpBIN35S-S.S-CAT and transformed into E. coli JM109. The resulting E.coli transformant was designated E. coli JM109/pBIN35S-S.S-CAT.

The plasmid with an expression cassette containing a peroxidase gene anda 35S promoter was prepared by the scheme as shown in FIG. 3. Plasmidvector pKK-PER1 was digested with Eco RI and Hin dIII so as to containthe open reading frame in the nucleotide sequence of SEQ ID NO: 3(prxC1a). This fragment was blunt-ended with a Klenow fragment andsubcloned into the Sma I site of plasmid pUC19. This plasmid wasdigested with Xba I and Sac I, and the fragment was subcloned betweenthe Xba I and Sac I sites of binary vector pBI121 having kanamycinresistance gene (Km) and a 35S promoter. This plasmid was designatedpBI35S-prxC1 and transformed into E. coli JM109. The resulting E. colitransformant was designated E. coli JM109/pBI35S-prxC1a.

2. Introduction of Each Plasmid into Agrobacterium

The transformant E. coli JM109/pBI35S-22, JM109/pBIN35S-S.S-CAT orJM109/pBI35S-prxC1a obtained in section 1 and E. coli strain HB101containing helper plasmid pRK2013 were separately cultured on LB mediacontaining 50 mg/l of kanamycin at 37° C. overnight, while theAgrobacterium strain EHA101 was cultured on LB medium containing 50 mg/lof kanamycin at 37° C. over two successive nights. The bacterial cellswere harvested by taking 1.5 ml of each culture in an Eppendorf tube,and then washed with LB medium. These bacterial cells were suspended in1 ml of LB medium, after which three kinds of bacteria were mixedtogether in 100 μl portions. The mixture was plated on LB agar mediumand incubated at 28° C. to ensure the conjugation transfer of plasmidsto Agrobacterium. After 1 to 2 days, a part of the medium was scratchedwith a sterile loop and spread over LB agar medium containing 50 mg/lkanamycin. The incubation was continued at 28° C. for 2 days, and asingle colony was selected. The resulting transformants were designatedEHA101/pBI35S-22, EHA101/pBIN35S-S.S-CAT and EHA101/pBI35S-prxC1a.

3. Preparation of Test Plants

(1) Seed Sterilization and Germination

Cotton seeds were soaked in 70% ethanol with stirring for 30 seconds.The seeds were then collected in a sieve and rinsed with 100 ml ofsterilized water. The seeds were then soaked in Clorox/Tween 20 solutionand shaken at 110 rpm for 20 minutes. The seeds were collected in asieve and rinsed with 100 ml of sterilized water. The seeds were soakedin sterilized water with shaking for 2 minutes and collected in a sieve.The rinsing steps were repeated twice, and the seeds were finally soakedin sterilized water for 30 to 60 minutes. The seeds were collected in asieve and placed on germination media (100 ml Stewart'sconcentrate/liter, pH 6.8 with 1 M KOH, 2.0 g/l Phytagel (Sigma)) in25×150 mm culture tubes and incubated at 30° C. for 7 to 10 days.

(2) Hypocotyl Inoculation

Two milliliter cultures of the Agrobacterium containing the geneconstructs were grown in the presence of appropriate antibiotics at 30°C. for 24 hours. The cultures were diluted in the ratio of 19:1 withMSNH (MS basal salts (Sigma), 30 g/l glucose, pH 5.8) in a sterile Petridish. Cotton seedlings (7 to 10 days after sowing) were removed from theculture tubes and placed on a sterile aluminum foil cutting pad.Cotyledons and roots were excised and discarded, and the hypocotyls werecut into pieces of 6 to 8 mm in length. The hypocotyls were placed inthe diluted Agrobacterium cultures and incubated for at least 30seconds. The hypocotyl pieces were removed and blotted on sterile filterpaper to remove excess Agrobacterium solution. The hypocotyl pieces wereplaced on Petri dishes containing T₂ media (MS basal salts (Sigma), 0.75g/l MgCl₂, 0.1 mg/l 2,4-D, 0.5 mg/l kinetin, 30 g/l glucose, 2.0 g/lPhytagel (Sigma), pH 5.8) at a ratio of 8 to 10 hypocotyl pieces perdish. Approximately 200 to 300 hypocotyl pieces (20 to 30 plates) wereused for each inoculation. The dishes that contained the inoculationhypocotyls were incubated in the dark at room temperature for 3 days.The hypocotyls were removed from the T₂ plates, blotted on sterilefilter paper, and transferred to MS2NK KCL plates (MS basal salts(Sigma), 0.75 g/l MgCl₂, 30 g/l glucose, 2 mg/l NAA, 0.1 mg/l kinetin,2.0 g/l Phytagel (Sigma), 1 ml of 50 mg/ml kanamycin/liter, 5 ml of 100mg/ml cefotaxime/liter, pH 5.8; 45 to 50 ml of media per Petri dish).These plates were sealed with Parafilm to prevent drying. These platesincubated under the light at 30° C.

(3) Callus Growth

The hypocotyls incubated on MS2NK KCL plates were transferred once permonth or as needed to avoid contamination and overgrowth of theAgrobacterium. The transformed callus began to appear in 3 to 4 weeks.The transformed callus was light green or white in color, and appearedto be smooth and dense rather than loose and fluffy.

When the calluses were approximately 4 mm in size, they were removedfrom the hypocotyl and placed on separate MS2NK KCL plates to encouragefaster growth. When the individual calluses are approximately 1 cm insize, they were transferred into suspension culture to begin generationof embryos. The time required to reach this stage of growth wastypically 3 to 5 months.

(4) Embryo Generation

The calluses were placed in a 50 ml of Erlenmeyer flask containing 10 mlof MSNH and 5 μl of 50 mg/ml kanamycin. The calluses were broken intosmall pieces using a sterile spatula. Suspension cultures were incubatedon a shaker at 110 rpm in a lighted area at 30° C. The cultures wereready to plate in 2 to 8 weeks when the cells became light green incolor with some small, white slightly round cells visible in thecultures.

(5) Plating Suspension Cultures

The suspension cultures were poured into sterile 50 ml tubes and thecells were allowed to settle to the bottom of the tubes. The MSNH wasremoved and 20 to 30 ml of fresh MSNH was added to rinse the cells. Thetubes were shaken and the cells were allowed to settle to the bottomagain. This rinsing procedure was repeated twice.

After the cells settled to the bottom of the tubes, the cell volume wasestimated and the MSNH used to rinse the cells was removed and the cellsresuspended in MSNH so that they were diluted at a ratio of 0.5 ml ofcells in 9.5 ml of MSNH. Two milliliter aliquots of this suspension wasapplied to MSK 50K plates (MS basal salts (Sigma), 0.75 g/l MgCl₂, 1.9g/l KNO₃, 30 g/l glucose, 2.0 g/l Phytagel (Sigma), 1 ml of 50 mg/mlkanamycin/liter, pH 5.8), and spread evenly over the media surface. Theplates were incubated under the light at 30° C. until the surface of theplates were dry and then sealed with Parafilm.

(6) Embryo Selection

All of the plates were examined at least one each month for somaticembryo development. The somatic embryos became visible on some of theplates after about 3 weeks. When the embryos reached at least 1 mm insize, they were placed on SA plates (100 ml Stewart's concentrate/liter,20 g/l sucrose, 20 g/l agar, pH 6.8; pour about 20 ml in each Petridish) to begin the germination process. The other potentiallyembryogenic cells were removed to fresh MSK 50K plates. These cells wereusually light green rather than brown or white, and were round andglassy looking. If a particular cell line did not produce anyembryogenic cells, some of the green non-embryogenic cells were washedwith MSNH and replated as described above.

(7) Embryo Germination

After the embryos were selected from the MSK 50K plates, they wereplaced on SA plates and then incubated without Parafilm sealing to begindesiccation. The plates were incubated in the dark at 30° C. forapproximately 2 weeks. After two weeks, the roots were trimmed and theembryos were transferred to SGA plates (Stewart's concentrate, 5 g/lsucrose, 0.75 g/l MgCl₂, 1.5 g/l Phytagel (Sigma), 5 g/l agar, pH 6.8;pour about 30 ml in each Petri dish). The SGA plates were incubated inthe light at 30° C. without Parafilm sealing. The embryos weretransferred to new SGA plates as the old plates dried out, and the rootswere trimmed each time they were transferred. The germinated embryosremained on the SGA plates until true leaves emerged.

(8) Cotton Plants

After the first true leaves emerged, the young plantlets were moved topint sized canning jars containing SGA media (same as SGA plates, exceptthat approximately 60 ml of media should be added to each jar). When theplants reached 7 to 10 cm in height and had several true leaves, the topwas cut off and transferred to another jar. The cuttings had 3 to 4leaves. The parent plants were maintained in jars in case the cuttingsaccidentally died in the greenhouse.

When the cuttings developed a good root system, but before the rootsturned black, they were transplanted into one gallon pots containingpotting soil. The plants were hardened by placing large Zip-lock bagsover the plants and the top of the pot. After two weeks, the corners ofthe bag were cut off and left for one week. The top was then cutcompletely open and left or one more week. The bags were then removed,and the hardened plants ere moved to the greenhouse.

The Stewart's concentrate (10×) used herein was prepared from thefollowing materials:

Macronutrients KNO₃ 20.24 g NH₄NO₃  9.60 g MgSO₄.7H₂O 19.72 g CaCl₂.2H₂O 7.04 g KH₂PO₄  1.08 g Micronutrients Fe EDTA   220 mg Boric acid   74mg MnSO₄.4H₂O 202.8 mg ZnSO₄.7H₂O 103.6 mg KI   10 mg NaMoO₂. 2H₂O  8.8mg CuSO₄.5H₂O  0.3 mg CoCl₂.6H₂O 0.284 mg Vitamins Nicotinic acid  19.6mg Pyridoxine HCl  32.8 mg Thiamine HCl   54 mg

This will make 4 liters of concentrate, which is enough for 40 liters ofmedia. This concentrate was frozen in aliquots of 100 ml.

EXAMPLE 5 Field Test of Transformants

1. Selection of Cell Lines Having Desired Genes Introduced Therein

The transformants as prepared in Example 4 were subjected to thecultivation test in a closed greenhouse attached to Texas TechUniversity. The introduction of a desired gene was checked by Northernanalysis and the cotton fibers were evaluated for fiber characteristics.Cell lines having the desired genes surely introduced and expressed wereselected. The selected cell lines are shown in Table 1.

TABLE 1 Introduced gene Cell line KC22 7-114#5-34 KC22 114#41-3 KC22114#46-10 CAT R-9#15-1 CAT 9-52-16 prxC1a 11#27-31-1 prxC1a 11#14-12CprxC1a 11#14-12A

2. Cultivation Test in Separated Field

The seeds of the cell lines shown in Table 1 and a control cotton plant(Coker 312) were used as the test samples. These seeds were sown in aseparated field. These cotton plants began flowering, at which time theyunderwent self-pollination. In a few subsequent months, young healthyleaves were collected from all the transformants under cultivation, andexamined for the expression of the desired genes introduced therein byNorthern analysis using these genes as probes according to the ordinarymethod. After opening, cotton bolls were collected and dried. Seedcotton was collected and subjected to machine ginning in the Texas A&MAgricultural Experiment Station to separate cotton fibers from theseeds. The cotton fibers were evaluated for fiber characteristics suchas (1) fiber length by the sorter method and fiber strength by thepressley method; and (2) fiber length, fiber strength and fiber finenessby 900 HVI system (Spinlab). The results are shown in Tables 2 and 3 forfiber characteristics (1) and (2), respectively.

TABLE 2 Introduced Fiber length Fiber strength gene Cell line (inch)(1000 Lbs/in²) non- Coker 312 1.21 ± 0.03 86.4 ± 2.6 transformant KC227-114#5-34 1.31 ± 0.03 91.0 ± 3.2 KC22 114#41-3 1.35 ± 0.02 87.9 ± 3.3KC22 114#46-10 1.32 ± 0.03 90.5 ± 2.4 CAT R-9#15-1 1.20 ± 0.03 95.0 ±4.4 CAT 9-52-16 1.30 ± 0.03 95.3 ± 5.1 prxC1a 11#27-31-1 1.26 ± 0.0490.9 ± 4.1 prxC1a 11#14-12C 1.28 ± 0.04 88.7 ± 3.2 prxC1a 11#14-12A 1.31± 0.02 88.6 ± 1.5

TABLE 3 Fiber Fiber Fiber Introduced length strength fineness gene Cellline (inch) (g/tex) (μg/inch) non- Coker 312 1.15 ± 0.04 24.2 ± 1.7 4.00± 0.54 transformant KC22 7-114#5-34 1.29 ± 0.02 26.9 ± 1.0 4.80 ± 0.10KC22 114#41-3 1.24 ± 0.01 27.5 ± 0.4 4.90 ± 0.10 KC22 114#46-10 1.28 ±0.02 28.0 ± 0.8 4.65 ± 0.07 CAT R-9#15-1 1.15 ± 0.03 28.9 ± 1.2 5.05 ±0.21 CAT 9-52-16 1.25 ± 0.02 28.2 ± 0.6 4.80 ± 0.30 prxC1a 11#27-31-11.25 ± 0.01 28.2 ± 1.2 4.10 ± 0.10 prxC1a 11#14-12C 1.26 ± 0.02 26.1 ±1.2 4.33 ± 0.26 prxC1a 11#14-12A 1.28 ± 0.02 25.6 ± 1.5 4.15 ± 0.07

As can be seen from Tables 2 and 3, all the cell lines (i.e.,7-114#5-34, 114#41-3 and 114#46-10) of the transformants having cottonplant endoxyloglucan transferase gene (KC22) introduced thereinexhibited a remarkable increase both in fiber length and in fiberfineness. In addition, all the cell lines (i.e., R-9#15-1 and 9-52-16)of the transformants having pea catalase gene (CAT) introduced thereinexhibited a remarkable increase both in fiber strength and in fiberfineness, whereas all the cell lines (i.e., 11#27-31-1, 11#14-12C and11#14-12A) of the transformants having horseradish peroxidase gene(prxC1a) introduced therein exhibits a remarkable increase in fiberlength.

These findings show that the introduction of a gene coding for such aspecific enzyme in cotton plants can attain a significant improvement offiber characteristics, e.g., fiber length and fiber fineness in the caseof cotton plant endoxyloglucan transferase gene (KC22); fiber strengthand fiber fineness in the case of pea catalase gene (CAT); and fiberlength in the case of horseradish peroxidase gene (prxC1a).

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 3 <210> SEQ ID NO 1 <211>LENGTH: 1035 <212> TYPE: DNA <213> ORGANISM: Gossypium barbadense <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (32)...(901) <300>PUBLICATION INFORMATION: <310> PATENT DOCUMENT NUMBER: US, A, 5880110<311> PATENT FILING DATE: 1995-02-21 <312> PUBLICATION DATE: 1999-03-09<313> RELEVANT RESIDUES: 1, (1)...(1035) <400> SEQUENCE: 1 caataattctctctgtttct ctggtttaaa catgggtatg ggtttaagga atggatttct 60 tttgattttatcttgtgttg ttacactttc cctctcagtt ttggggcgac ctgccacttt 120 ccttgaagattttagaatca cttggtctga ttctcatatt aggcaaatcg atggagggag 180 agccatccaacttgttctcg accaaaattc aggctgtgga tttgcttcta aaaggcagta 240 tttgttcggacgtgtcagca tgaaaatcaa gctcatcccc ggcgactccg ccggaacagt 300 caccgccttttatatgaatt ctgttacaga tgctgtgcga gatgagctag acttcgagtt 360 cttgggaaaccgtaccgggc agccatatac ggttcaaacc aatatctatg cccatggaaa 420 gggtgacagggaacaaaggg ttaacctttg gttcgatcct gctgcagatt tccatactta 480 ctcaatcatgtggaaccatc atcagattgt gttctatatt gatgaagtgc caattagggt 540 ttataagaacaatgaagcta gaaatatccc atacccaaaa ctccagccaa tgggagttta 600 ttcaacgctgtgggaggctg atgattgggc aacaagggga ggtttagaga aaattgattg 660 gaccaaagctccgttcttag cttattacaa ggacttcgac attgaaggat gtccggttcc 720 agggccagtaaactgtgcca caaacagtag gaactggtgg gagggcactg cttatcaagc 780 ccttaatgccatggaagcta aaagatatag ttgggttcgt atgaaccacg tgatatacga 840 ttactgcaccgacaagtccc gttacccggt taccccaccg gagtgcatgt ccatcatctg 900 aaaatccaaacccaagtgaa gtttcgtgtc ctattttacg tacatatgta cctcccttta 960 tacaaataatagagccatgc aaaaattggg ttttaaaaaa aaaaaaaaaa aaaaaaaaaa 1020 aaaaaaaaaaaaaaa 1035 <210> SEQ ID NO 2 <211> LENGTH: 1738 <212> TYPE: DNA <213>ORGANISM: Pisum sativum <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (57)...(1541) <300> PUBLICATION INFORMATION: <301> AUTHORS:Sibel H. Isin and Randy D. Allen <302> TITLE: Isolation andcharacterization of a pea catalase cDNA <303> JOURNAL: Plant MolecularBiology <304> VOLUME: 17 <306> PAGES: 1263-1265 <307> DATE: 1991 <400>SEQUENCE: 2 attttctcta atccctatct tctgctccac caccaccgtc tatcgcttccatttccatgg 60 atccttacaa gcatcgtcct tctagcgctt tcaattctcc tttctggactacgaactccg 120 gtgctcctgt ttggaataat aactcttccc taaccgttgg atctagaggtccaattctat 180 tggaagatta tcatcttgtg gaaaagcttg cccaatttga tagggaaaggatcccagaac 240 gtgttgtcca tgctagggga gcaagtgcaa agggtttctt tgaagtcacacatgatattt 300 cgcacctgac atgtgcagat ttccttcgag cccctggtgt tcagacacctgtcattgtgc 360 gtttctcaac tgtcattcat gaacgtggca gccctgaaac cttgagggatccccgaggtt 420 ttgctgtgaa attttacacc agagagggta actatgacct tgttggaaacaactttcccg 480 tcttcttcgt tcatgacggt atgaattttc cagatatggt ccatgctcttaaacccaatc 540 cccagaccca catccaggag aattggagaa ttcttgattt cttctacaactttccagaaa 600 gccttcacat gttctccttc ctatttgatg atgtgggtgt cccacaagattataggcata 660 tggatggttt tggagttaac acatacaccc tgatcaacaa ggctggaaaatcggtgtatg 720 tcaaatttca ctggaagccc acctgtggtg tgaagtgtct attggaggaagaggccattc 780 aggtgggagg atccaaccac agccatgcta ctaaagacct ttatgactcaattgctgctg 840 gtaactatcc tgagtggaaa ctttacattc aaacaataga tcctgctcatgaagacagat 900 ttgagtttga cccacttgat gtaactaaga cttggcccga ggacatcataccccttcagc 960 ccgtaggtcg catggtcttg aacaagaaca tagataattt ctttgctgagaatgaacagc 1020 ttgcattttg tcctgccatt atgctgcctg gtatatatta ctcagatgacaagatgcttc 1080 aaactagggt tttctcttat gctgattcac agaggcacag acttggaccgaactacctgc 1140 aacttcctgt taatgctccc aagtggtctc accacaacaa tcaccatgagggtttcatga 1200 atgccattca cagggatgag gaggtcaatt acttcccttc aaggcatgatactgttcgtc 1260 atgcagaaag ggtccccatt cctactactc atttatctgc aaggcgtgaaaagtgcaata 1320 ttccgaaaca gaatcacttc aagcaggctg gagaaagata ccgaacttgggcacctgaca 1380 ggcaggaaag atttctccgc aggtgggtag aagctttatc cgacaccgatccacgcatca 1440 cccatgaaat ccgcagcatt tgggtatcat actggtctca ggctgatcgttctcttgggc 1500 agaagttagc atctcatctg aacatgaggc ctagcattta actttgttgccaaatattga 1560 atcatcgcaa gatttgcaga tgtgcaaaat gtatgataaa ggatgtttgtttggattact 1620 tgaaaagact ttttattttt gttataattt tatatcgtga atgtataccataaattctat 1680 gtatgcaact cgttgagatg ttacaataaa tccgtaggca tgtgttagtgttaaaaaa 1738 <210> SEQ ID NO 3 <211> LENGTH: 1062 <212> TYPE: DNA <213>ORGANISM: Armoracia rusticana <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)...(1062) <300> PUBLICATION INFORMATION: <301> AUTHORS:Kazuhito FUJIYAMA et al. <302> TITLE: Structure of the horseradishperoxidase isozyme C genes <303> JOURNAL: European Journal ofBiochemistry <304> VOLUME: 173 <306> PAGES: 681-687 <307> DATE: 1988<400> SEQUENCE: 3 atgcatttct cttcttcttc tactttgttc acttgtataa ccttaatcccattggtatgt 60 cttattcttc atgcttcttt gtctgatgct caacttaccc ctaccttcatcgacaattca 120 tgtcctaatg tctctaacat cgtacgggat actattgtca atgagctaagatcagaccct 180 cgtattgccg cgagcatcct tcgtcttcac ttccacgact gctttgttaatggttgtgac 240 gcatcgatct tgttagacaa cacaacatca tttcgaacag agaaagatgcgtttggaaac 300 gcaaactcgg caagaggatt tccagtgatt gatagaatga aagccgcggtggagagtgca 360 tgcccaagaa ccgtttcatg cgcagatttg ctcaccattg cagctcaacaatctgtcact 420 ttggcgggag gtccttcttg gagagttcct ttgggcagaa gagatagcttacaagcattt 480 ctggatcttg ctaatgcaaa tcttccagct ccattcttca cacttccacaacttaaagac 540 agctttagaa atgttggcct caaccgttct tctgatctcg ttgcactgtccgggggccac 600 acatttggta aaaatcagtg tcggtttatt atggacagat tatacaacttcagcaacacc 660 ggtttacccg atcctactct caacactact tatctccaaa ctcttcgtggactatgtccc 720 ctcaatggta atctaagcgc tttggtggat tttgatctac gtacgccaacgatttttgac 780 aacaaatact atgtgaatct cgaagagcaa aaaggactta tccaaagcgaccaagagttg 840 ttctctagcc ccaatgccac tgacacaatc cctttggtga gatcatttgctaatagcaca 900 caaacattct tcaatgcgtt tgtggaggcg atggatagga tgggaaacattacacctctt 960 acaggaactc aaggacagat caggttgaat tgtagggtgg tgaactccaactctctactc 1020 catgatatgg tggaggtcgt tgactttgtt agctctatgt ga 1062

What is claimed is:
 1. A cotton plant of the genus Gossypium with improved cotton fiber characteristics, comprising an expression cassette containing a polynucleotide coding for peroxidase operably linked to a promoter that directs expression in cotton fiber cells, so that the polynucleotide is expressed in cotton fiber cells to improve fiber length, fiber fineness, or fiber strength as compared to cotton fiber cells from a non-transformed cotton plant.
 2. The cotton plant according to claim 1, wherein the polynucleotide coding for peroxidase is derived from a plant.
 3. The cotton plant according to claim 1, wherein the polynucleotide coding for peroxidase has a nucleotide sequence extending from bps 1 to 1062 of SEQ ID NO:3.
 4. The cotton plant according to claim 1, which is selected from the group consisting of Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, Gossypium anomalum, Gossypium armourianum, Gossypium klotzchianum, Gossypium herbaceum and Gossypium raimondii.
 5. Cotton fibers obtained from the cotton plant according to claim
 1. 6. Cotton seeds obtained from the cotton plant according to claim 1, wherein the cotton seeds comprise the expression cassette of claim
 1. 7. A method for producing cotton fibers with improved fiber characteristics, which comprises the steps of: transforming into a cotton plant of the genus Gossypium, an expression cassette containing a polynucleotide coding for peroxidase operably linked to a promoter that directs expression in cotton fiber cells, so that the gene is expressed in cotton fiber cells to improve fiber length, fiber fineness, or fiber strength as compared to cotton fiber cells from a non-transformed cotton plant; growing the cotton plant transforment to form cotton bolls; and collecting cotton fibers from the cotton bolls of the plant.
 8. The method according to claim 7, wherein the polynucleotide coding for peroxidase is derived from a plant.
 9. The method according to claim 8, wherein the polynucleotide coding for peroxidase is derived from a plant.
 10. The method according claim 7, wherein the polynucleotide coding for peroxidase has a nucleotide sequence extending from bps 1 to 1062 of SEQ ID NO:
 3. 11. The method according to claim 7, wherein the cotton plant of the genus Gossypium is selected from the group consisting of Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, Gossypium anomalum, Gossypium armourianum, Gossypium klotzchianum, Gossypium herbaceum and Gossypium raimondii.
 12. Cotton fibers obtained from the method according to claim
 7. 